Anti-proliferative preparations

ABSTRACT

A dormant preparation (DC) is provided which is capable of inhibiting proliferation of various kinds of cells. The preparation comprises an extract which is obtained from cells or tissue originating in an organism capable of entering a phase of dormancy in at least one of its parts and comprises at least one substance which induces or maintains the state of dormancy in the organism from which the cells or tissue are derived. The DC may be used for a variety of indications including human medicine and cosmetics, plant growth control and food preservation. A preferred dormant preparation is prepared from a water extract of Narcissus (daffodil) bulb.

FIELD OF THE INVENTION

The present invention concerns extracts of cells or tissue or theirsupernatants which can inhibit proliferation of cells or tissue. Theinvention also provides compositions comprising such extracts as well aspharmaceutical, cosmetic and agricultural uses of the compositions andextracts.

BACKGROUND OF THE INVENTION

Dormancy is a phenomena which is found in representatives of the plantkingdom as well as the animal kingdom.

The germination of various grains and seeds comprising the necessarypropagation organs is delayed under certain circumstances and yet thegrains or seeds are capable of germinating after various periods oftime. The period of time in which germination of such seeds may bedelayed varies and depends both on intrinsic properties of the seed aswell as on the nature and extremity of the environmental conditions.Seeds have been shown to be in dormancy for a few days, a year, severalyears and even for more than several centuries (as was discovered latelyin the case of some nympheaceae and seeds of trees of the Leguminosaefamily (Shen-Miller, J., et al., American Journal of Botany.82:1367-1380, 1995)).

In some cases, the capability to develop dormancy lies in the embryoenvelopes. In such a case, the separation of the envelopes from theembryo, result in its immediate germination.

In other cases, chemical growth inhibitors capable of preventinggermination are present in the embryo itself and thus even a bare embryomay remain dormant (such as in the case of Rosaceae plants such asKerria, Peach, etc.).

In plants, a state of dormancy may be found in the whole plant or in oneor more of its parts. Dormant plants are plants which have two mainmetabolic states in their growth cycle. In their dormant state, theplants' metabolism is extremely low, and the plant growth process issignificantly inhibited although differentiation of certain cells mayoccur. In their active state, the plants' metabolism rate is higher, thecells divide and differentiate and there is significant growth ofvarious parts of the plant. In some cases, the whole plant enters thedormant state. Such is the case in Narcissus plants in which during thedormant state the only remaining viable part is the bulb which is in itsdormant state. In other cases, some parts of the plants may be activewhile other parts may be in dormancy such as, for example, is the caseof apple trees.

Substances capable of inhibiting germination have also been shown to bepresent in the juice of fleshy fruits or in other plant organs whichproduce juice. Examples are tomatoes, grapes, kiwi, watermelon andgrapefruit wherein pips present in the fruit do not germinate althoughtheir surroundings are suitable for germination due to the water withinthe fruit.

Several plant-derived substances having an effect on cell proliferationhave been reported. For example, European Patent Application No. 0381514describes compositions comprising both naturally derived as well assynthetically prepared sphingolipids which have growth inhibitoryactivity on various kinds of cells. Another well known plant-derivedsubstance having an anti-mitotic effect on various kinds of human cellsis the substance colchicine (Samson, F. E., A. Rev. Pharmac. Toxic16:143 (1976)). The Narcissus alkaloid, pretazettine, was shown to havea cytotoxic effect on Rausher virus-carrier cells as well asanti-leukemic activity in leukemic mice although the predominantactivity of the substance was shown to be an antiviral activity(Furusawa, E. et al., Chemotherapy, 26:36-45, (1980) and Furusawa, E. etal., Proc. Soc. Exp. Biol. Med. 152:186-191, (1976)). Utex europaezisseed extracts were shown to comprise a non glycoprotein lectin capableof reversibly inhibiting growth of certain lymphocytes as well as toinhibit the growth of various reticulo endothelial tumor cell lines(Pirofsky, B., et aL, Vox-Sano, 42:295-303, (1982) and Pirofsky, B., etal., J. Biol. Response Mod., 2:175-185, (1983)). Root extract of Panexginseng was shown to decrease DNA synthesis measured by [H³]-thymidineincorporation of V 79 Chinese hampster lung cells. Another substance,Narciclasine obtained from bulbs of various Narcissus varieties wasshown, amongst other of its activities, to inhibit growth of wheat kemalradicals (Ceriotti, G., et al, Tumors 53:359-371 (1967)). Bulbs ofPancratium littoral collected in Hawaii were found to contain a productdesignated pancratistatin capable of inhibiting growth of variousneoplastic cell lines in vitro (Pettit, G. R., et al., J. Nat. Prod.,49:995-1002 (1986)).

Against this, many plant extracts having an opposite effect on cells,i.e., capable of augmenting their proliferation were also described suchas, for example, the methanolic extract from the root of Scutellariabaicalensis georgi were shown to significantly augment the cellularactivity of fibroblasts (Chung, C.P., et al, Planta-Med, 61:150-153,(1995)). Gibberellin-like growth substances were found in six differentplant species having bulbs (Staby, G. L., Hort. Science, 399-400(1970)). Several cytokinins which were found in roots that developedfrom Narcissus bulbs had an effect on bulb growth of the plants in whichthey were detected (Vanstaden, J. V., Pflanzenphysiol., 86:323-30(1978)).

The phenomena of dormancy may also be found in the animal kingdom, forexample, in the small crustacean Artemia salina (Finamore and Clegg Ln:The Cell Cycle, Academic Press Ed., 249-278, 1969). The naturalenvironment of this marine crustacea is usually briny ponds. Afterfertilization, the early stages of development of artemia involve theformation of a blastula which then becomes a gastrula. Under severeenvironmental conditions such as dehydration (drought), the gastrula iscapable of forming a cyst wherein the whole organism enters a dormancyphase. The dormant artemia gastrula (commonly miscalled “artemia eggs”)are capable of remaining in their dormant state for many years. When theencysted gastrula are rehydrated, the various metabolic activities ofthe artemia are resumed and protein synthesis can be seen after about 10minutes. However, DNA synthesis and cell division are absent until aboutafter 60 hours (Le Gal, Y, In: Biochinie Marine, (Ed. Masson) p. 176,1988).

Various plant derived compositions (such as retinoic acid U.S. Pat. No.5,438,073) and α-hydroxy acids (Ditre, C. M., et al., J. Am. AcadDermatol., 34:187-195, 1996)) as well as animal derived extracts havebeen proposed for use in the cosmetic field for stimulating theproliferation and renewal of epidermal cells. Such compositions wereconsidered to be useful in the cosmetic field where it is accepted thatthe natural renewal process of epidermis is slowed down with aging. Itis believed that removal of the outer surface with simultaneousstimulation of growth of new cells in the inner layers of the epidermisto divide and migrate to the outer surface, will result in skin renewaland in a younger skin appearance. However, it is also known and has beenrecently shown that the increase in cell division is a crucial factor inconverting normal cells into premalignant or malignant cells (Ames, B.N. et al, Environ. Health Perspect101:35-44 (1993)).

It is also believed today that normal human and animal cells have afinite capacity to replicate. It has been shown that the number ofmitotic events that cultured normal animal cells can undergo appear tobe inversely related to the age of the donor from which they wereobtained (Hayflick, L., Clin. Geriatr. Med., 1:15-27, (1985)). It hasalso been shown that cell cultures obtained from patients withaccelerated aging syndromes undergo less replications than cell culturesobtained from age matched control individuals.

GLOSSARY

The following is the meaning of some terms of which will be used in thetext below:

Dormancy—a state in which there is a marked decrease in the metabolicrate of cells or tissues resulting in the inhibition of growth andproliferation of the cells or tissue.

Dormans—substances naturally found in cells or tissue and which arecapable of inducing cells or tissue to enter a state of dormancy or ofmaintaining the dormant state in cells or tissues that have alreadyentered that state. The dormans may be obtained from a variety of plantparts which are capable of entering into a state of dormancy; from juiceof various fruits which contain dormans capable of inhibitinggermination of seeds within the fruit; from animals which are capable ofentering a phase of dormancy in their life cycle, e.g. gastrula ofcertain crustaceans such as artemia or dafnia; etc. In some cases, thedormans are found within a dormant tissue, e.g. in a dormant seed or inthe gastrula of artemia or dafnia; in other cases the dormans are foundin a tissue surrounding the dorman tissue or organ, e.g. in a fruitjuice surrounding a dormant seed.

Extract—At least one substance obtained by any of a variety ofextraction methods known in the art. For example, the extract may be anaqueous extract, a glycolic extract an alcoholic extract, an oilyextract, etc. The extract in accordance with the invention is obtainedfrom cells or tissues from a part of a plant or animal capable ofentering a state of dormancy. The cells or tissue may be obtaineddirectly from the plant or animal and the extract may then be preparedtherefrom. Alternatively, cell cultures may first be prepared from theplant or animal cells or tissues and then the cell cultures may be grownfor various periods of time. In order to prepare an extract, the cellsare then harvested from the cell cultures, the cells and their growthmedium are separated and an extract may be prepared either from thecells themselves or from the growth medium (which will be referred to as“supernatant”) which contains substances secreted by the cells intotheir growth medium. Thus, the “extract ” may be obtained directly fromplant or animal tissue or from an animal or plant cell or tissueculture.

Dorman extract—an extract obtained from a plant cell or tissue, fromfruit or from an animal cell or tissue which comprises dormans.

Enriched dorman preparation (EDP)—a preparation derived from a naturalsource which comprises dormans in a concentration larger than that whichis found in a natural unprocessed extract. The EDP may be obtained bypurification of a natural extract to obtain fractions which containeddormans in the larger concentration, e.g. by various chromatographictechniques, by filtration, etc., as well as by biological meansincluding growing cells or tissue which are capable of producing dormansunder conditions in which they produce dormans in relatively largequantities and collecting their secretion products. In order todetermine whether a preparation is an enriched dorman preparation, thepreparation may be assayed for a specific biological activity associatedwith dormans, as described below. EDP contains a substantially higherconcentration of dormans as compared to the natural preparation, e.g. atleast 1.5 folds, preferably 2 folds and typically at least 2.5 folds tothe concentration of the dormans in the natural preparation.

Producer cells or producer tissue—cells or tissue which are capable ofproducing dormans which may thus be extracted therefrom.

Target cells or target tissue—cells or tissue which are contacted withdormans, in accordance with the invention and which thereby enter astate of inhibition of their growth or proliferation or maintain such astate as the result of contact with dormans.

Dorman analog—a substance, typically synthetic, which has a dorman-likeactivity in that it is capable of inducing dormancy in the same cells ortissue induced to dormancy by the dorman and which, in accordance withthe invention, is also capable of inhibiting growth and proliferatin oftarget cells or tissue.

Dorman composition (DC)—a composition comprising, as active ingredient,an amount of dorman (e.g. as a dorman extract) or dorman analogeffective in inhibiting growth and proliferation of target cells ortissue (“effective amount”). A dorman composition may comprise anaturally derived EDP, a composition comprising synthetic dormans aswell as dorman analogs.

Active Extract (AE)—extracts obtained from cells or tissues from a partof a plant or animal capable of entering a state of dormancy during thenon dormant state.

SUMMARY OF THE INVENTION

In accordance with the invention, use is made of a dorman composition.The composition may be used for inhibiting proliferation of cells,particularly cells xenogeneic to the producer cells or tissue. By onepreferred embodiment of the invention, the compositions are used inhuman medicine and cosmetics. In accordance with another embodiment, thecompositions are used for controlling plant growth. By yet a furtherembodiment, the compositions of the invention are used in foodpreservation.

By one aspect of the invention there is thus provided a preparationcapable of inhibiting proliferation of target cells or target tissues,comprising a member selected from the group consisting of:

(i) an extract obtained from producer cells or producer tissue, saidcells or tissue originating in an organism capable of entering a phaseof dormancy in at least one of its parts, said extract comprising atleast one substance which induces or maintains said state of dormancy inan organism from which said producer cells or tissues are derived,

(ii) a dormancy inducing fraction of said extract; and

(iii) a dormancy inducing substance derived from said extract orfraction.

By one embodiment the composition comprises an enriched dormanpreparation (EDP) which, as defined above, comprises dormans in aconcentration larger than that which is found in a natural unprocessedextract.

The producer cells or tissues from which the extract is obtained may beof the same origin as said target cells or tissues but are preferably ofa different origin. In accordance with one embodiment of the invention,said target cells or tissue are human cells or tissue and said producercells or tissue are plant or non human animal cells or tissue. Inaccordance with another embodiment of the invention, said target cellsor tissue are plant cells or tissue.

In accordance with one preferred embodiment of the invention, the dormancomposition is a pharmaceutical or cosmetic composition for inhibitingcell proliferation within the body. In accordance with anotherembodiment of the invention, said composition is used for inhibitinggermination of seeds (being either natural or artificially prepared) orgrowth of seedlings for the purpose of maintaining seedlings in adormant state for example during storage. In accordance with a furtherembodiment of the invention, the dorman composition is used in freshfood preservation.

By one embodiment, the dormans or the extracts used in accordance withthe invention are derived from dormant plants.

Compositions in accordance with the invention obtained from bulbs ofdormant plants while in their dormant state are capable of inhibitingthe growth of seedlings as well as to inhibit the proliferation ofvarious target cells, including various mammalian cells, e.g. humancells, to a significantly higher extent than preparations obtained underthe same conditions from bulbs of the same plan being in their activestate. It was also found that dorman compositions obtained from cellcultures prepared from various parts of dormant plants and induced intodormancy are also capable of inhibiting the growth of seedlings as wellas to inhibit the proliferation of various cells, including variousmammalian cells, e.g. human cells. These DCs, in a wide range ofconcentrations, had no noticeable toxic effect on the target cells, thisbeing in contrast to most substances which have an anti-proliferatingeffect.

In accordance with the invention, any plant capable of entering adormant state may be used for obtaining an DC. Some non-limitingexamples of such plants, as well as the parts of such plants which entera dormant state (designated “D-part”) and from which the DC may beobtained, are shown in the following Tables I and II:

TABLE I (Bulbs, corms, roots, rhizomes) Name Family D-part AlliumLiliacease bulbs Amaryllis belladona Amaryllidacea bulbs AnemoneRanunculacea perennials & tuberous or fibrous roots Babiana Iridaceacorms Brodiaea Liliaceae corms Chionodoxa Liliaceae bulbs CrocusIridacea corms Ornithogallum Liliaceae bulbs Cyclamen Primulaceaeperennials & tuberous roots Endymion Liliaceae bulbs Arum Araceaeperennials & Tuberous roots Freesia Iridacea corms Fritillaria Liliaceaebulbs Galanthus Amaryllidacea bulbs Hippeastrum Amaryllidacea bulbsHyancinthus Liliaceae bulbs Leek Liliaceae bulbs Ipheion uniflorumAmaryllidacea bulbs Iris Iridacea bulbs & rhizomes Ixia Iridacea cormsLeucojum Amaryllidacea bulbs Lilium Liliaceae bulbs Muscari Liliaceaebulbs Narcissus Amaryllidacea bulbs Oxalis Oxalidaceae bulbs & rhizomesPaeonia Paeoniaceae tuberous perennials Puschkinia scilloides Liliaceaebulbs Ranuculus Ranunculacea tubers or perennials RadohypoxisHypoxidaceae bulbs Rhodophiala Amaryllidacea bulbs Scilia Liliaceaebulbs Sparaxis Iridacea corms Raritimum Liliaceae bulbs TriteleiaAmaryllidacea corms Tulipa Liliaceae bulbs Tritonia crocata Iridaceacorms Watsonia pyramidata Iridacea corms Zantedeschia Araceae rhizomesBegonia Begoniceae rhizomes Caladium Araceae perennials & tuberous rootsCanna Cannaceae perennials & tuberous roots Crocosmia Iridacea cormsDahlia Asteraceae perennials & tuberous roots Gladiolus Iridacea cormsGloriosa rothschildiana Liliaceae perennials & tuberous roots Homeriacollina Iridacea corms Hymenocallis Amaryllidacea bulbs LiatrisAsteraceae perennials Polianthes tuberosa Agavaceae perennials &tuberous roots Tigridia pavonia Iridacea bulbs Zantedeschia Araceaerhizomes Zephyranthes Amaryllidacea bulbs Colchicum Liliaceae cormsLycoris Amaryllidacea bulbs Sterenbergia lutea Amaryllidacea bulbsPancratium Liliaceae bulbs

TABLE II (Deciduous fruit trees, shrubs, seeds) Name Family D-partMalus - crabapple Malus Deciduous & fruit trees & shrubs MangiferaAnacardiaceae Deciduous & fruit trees & shrubs Peach Rosaceae Deciduous& fruit trees & shrubs Persimmon Ebenaceae Deciduous & fruit trees &shrubs Pistacia chinensis Anacardiaceae Deciduous & fruit trees & shrubsPrunus Rosaceae Deciduous & fruit trees & shrubs Fraxinus OleaceaeDeciduous & fruit trees & shrubs Pyrus Rosaceae Deciduous & fruit trees& shrubs Quercus Fagaceae Deciduous & fruit trees & shrubs SalixSalicaceae Deciduous & fruit trees & shrubs Actinidia ActinidiaceaeDeciduous & fruit trees & shrubs Akebia quinata LardizabalaceaeDeciduous & fruit trees & shrubs Blueberry Vitaceae Deciduous & fruittrees & shrubs Apple Rosaceae Deciduous & fruit trees & shrubs AloysiaVerbenaceae Deciduous & fruit trees & shrubs Campsis BignoniaceaeDeciduous & fruit trees & shrubs Celastrus Celastraceae Deciduous &fruit trees & shrubs Cellery Apiaceae seeds Clematis RanunculaceaeDeciduous & fruit trees & shrubs Grape Vitaceae Deciduous & fruit trees& shrubs Humulus Cannabaceae Deciduous & fruit trees & shrubs FigMoraceae Deciduous & fruit trees & shrubs Wisteria Fabaceae Deciduous &fruit trees & shrubs Bean Fabaceae Deciduous & fruit trees & shrubsLathyrus pea Fabaceae Deciduous & fruit trees & shrubs TropaeolumTropaeolaceae Deciduous & fruit trees & shrubs Amelanchier RosaceaeDeciduous & fruit trees & shrubs Cotoneaster Rosaceae Deciduous & fruittrees & shrubs Barberry Berberadaceae Deciduous & fruit trees & shrubsEnkianthus Ericaeae Deciduous & fruit trees & shrubs EunymusCelastraceae Deciduous & fruit trees & shrubs Kerria japonica RosaceaeDedicuous & fruit trees & shrubs Parsnip Apiaceae seeds PassifloraPassifloraceae Deciduous & fruit trees & shrubs Rhododendron EricaceaeDeciduous & fruit trees & shrubs Acacia Fabaceae Deciduous & fruit trees& shrubs Albizia Fabaceae Deciduous & fruit trees & shrubs AlmondRosaceae Deciduous & fruit trees & shrubs Ampelopsis Vitaceae veciduous& fruit trees & shrubs Anethum Apiaceae seeds Annona AnnonaceaeDeciduous & fruit trees & shrubs cherimola Apricot Rosacreae Deciduous &fruit trees & shrubs Artemisia Asteraceae Deciduous & fruit trees &shrubs Asparagus Liliaceae seeds Blackberry Rosaceae Deciduous & fruittrees & shrubs Carrot Apiaceae seeds Carya pecan Juglandeaceae Deciduous& fruit trees & shrubs Cherry Rosaceae Deciduous & fruit trees & shrubsCorn Poaceae seeds Helianthus Asteraceae seeds Cucumber Cucurbitaceaeseeds Filbert Betulaceae Deciduous & fruit trees & shrubs GooseberrySaxifragaceae Deciduous & fruit trees & shrubs Gourd CucurbitaceaeDeciduous & fruit trees & shrubs Lettuce Asteraceae seeds MelonCucurbitaceae seeds Okra Malvaceae seeds Onion Amaryllidaceae seeds orbulbs Peanut Fabaceae seeds Pear Rosaceae Deciduous & fruit trees &shrubs Pumpkin Cucurbitaceae seeds Punika garantium Punicaceae seedsRadish Cruciferae seeds Walnut Juglandeaceae Deciduous & fruit trees &shrubs Ziziphus jujuba Rhamanceae Deciduous & fruit trees & shrubsRaspberry Rosaceae Deciduous & fruit trees & shrubs Strawberry RosaceaeDeciduous & fruit trees & shrubs Turnip & Cruciferae Deciduous & fruittrees & shrubs rutabaga Malva Malvaceae seeds Verbascum Scrophulariaceaeseeds Chenopodium Chenopodiaceae seeds Nelumbo Nelumbonaceae seedsLupinus Papilonaceaee seeds

In accordance with the invention, DC is preferably obtained from plantswhich are in their dormant state either as a result of the naturalprocess of dormancy or as a result of being externally induced intodormancy by exposure which induce a dormant state e.g. conditions asincubation at a dormancy inducing temperature for a sufficient period oftime. The conditions for inducing dormancy in various dormant plants mayvary (e.g. the incubation temperature and the duration of theincubation) and are known to a person versed in the art. Thus forexample there are plants (such as Narcissus) induced into dormancy bytheir exposure to relatively high temperatures. Against this, otherplants (such as tulip) will be induced into dormancy by their exposureto relatively low temperatures. Other factors such as light, humidity,concentration of various growth factors, etc. may also be used to inducedormancy.

In accordance with one preferred embodiment of the invention, the DC isobtained from a part of the plant capable of entering dormancy (D-part),e.g. from bulbs. Bulbs induced into their dormant state may either beused immediately for the preparation of DC or, alternatively, may bestored under conditions which maintain a dormant state, e.g. in the caseof Narcissus these include high temperature and low humidity. Inaddition to bulbs, other parts of dormant plants such as combs, roots,seeds, etc., may also be induced into dormancy as explained above andthen used for obtaining DC therefrom.

In accordance with another embodiment of the invention, the dormanextract is obtained from cell cultures which were prepared from any partof a dormant plant (e.g. bulbs) and induced into dormancy. The culturemay be obtained by inoculation of bulbs of dormant plants havinginflorescence stalk initials into a suitable medium to form callouscultures of the bulb extracts. The cell cultures are then typicallygrown to confluency and very small bulb parts are formed in the cellculture (termed “bulblets”). Induction of a dormant state in the cellcultures or bulblets is obtained by their exposure to conditions whichinduce dormancy conditions, such as incubation at a dormancy inducingtemperature for a sufficient period of time. Dormancy may also beinduced in vitro by exposing the cell cultures or bulblets to varioustypes of chemical stresses (low or high concentrations of sugar, salts,etc.).

In addition to bulblets, cell cultures derived from other parts ofdormant plants such as combs, roots, seeds, etc., may also be used forobtaining cell culture derived dorman extracts.

By yet another embodiment, the dorman analogs of the invention may besynthetically prepared by any one of the methods known in the art suchas by recombinant DNA techniques, chemical synthesis, , combinationalchemistry, etc., the dorman analogs maintaining substantially similarcharacteristics as far as their ability to induce dormancy and inhibitproliferation of target cells of the dormans on which they are based.

Preferably, a plant derived DC of the invention is obtained as anaqueous extract of the plant material. The aqueous extract may beprepared by homogenizing the plant material and then suspending thehomogenate in an aqueous solution. However, non aqueous plant extractsobtained by any one of the extraction methods known per se, may also attimes be used in accordance with the invention.

In accordance with a further embodiment of the invention, the EDPs arcobtained from juice of fruits or other juice producing plant organs.Fruits typically contain dormans which inhibit germination of the seedsand pips while these are within the fruit. Examples of juices from whichdormans may be purified are juice of citrus fruits, grapes, tomato,kiwi, etc. The fruit juice may be used as such or alternatively, thedormans can be purified from the fruit juice as explained below.

In accordance with yet another embodiment of the invention, the dormansare extracted from producer cells originating from animals capable ofentering a dormant phase during their life cycle. During the dormantphase, the animals' metabolic rate is lowered to a minimum and there isan arrest in cell proliferation. Examples of such animals are variousmarine crustacea such as artemia, dafnia and cyclops.

As explained above with regards to plant derived DC, animal derived DCmay also be obtained from animals which are in their dormant state as aresult of the natural process of dormancy or as a result of beingexternally induced into dormancy by exposure to dormancy-inducingconditions such as dehydration (Artemia salina) or anoxia(Artemiafranciscana). The dormans may be extracted from the animaltissue by various methods known per se.

The animal derived DC may be obtained from the animals or their organsas such. Alternatively, cell cultures may first be prepared from thedormant animal, and after maintaining the cells in culture for variousperiods of time, DC may then be extracted either from the supernatant orby harvesting the cells and/or extracting the DC therefrom.

The DC of the invention may be purified from the producer cells by avariety of methods known per se, for example by chromatography (e.g.TLC, HPLC, ion exchange) by size fractionation (e.g. dialysis, gelfiltration), etc.

In accordance with the invention, it has been realized for the firsttime that, when administered to an individual, the anti-proliferativeactivity of said dorman composition may slow down the cell division rateof the cells present in the inner layers of the epidermis.

Another aspect of the invention is thus the use of said dormancompositions as a cosmetic or dermatological composition useful for talcmaintenance of the juvenile appearance of an individual's skin or forthe treatment of age related skin changes.

In accordance with this latter aspect of the present invention, adermatological or cosmetic composition is provided comprising from about0.0001%, preferably from about 0.001%, typically from about 0.01% up toabout 5% preferably up to about 1% by weight of dorman extract or dormananalog together with a dermatologically or cosmetically acceptablecarrier.

The dermatological or cosmetic compositions of the invention may beadministered in various forms such as in the form of a balm, anemulsified gel, an aqueous-alcoholic gel, anhydrous gel, an oil in water(O/W) type emulsion, a clear gel, cream containing liposomes, etc.

The cosmetic or dermatological compositions are typically topicallyadministered. However, it may at times be advantageous to administer thecompositions by other administration modes, such as, for example, bysubcutane injections, by orally administered capsules or byiontophoresis (which involves the use of electric fields to increase thepenetration of ionic active substances).

Due to their significant anti proliferative effect said dormancompositions may also be used for the treatment of various malignancies.As mentioned above, the cell division rate is a significant factor indetermining the probability of a cell to become a premalignant ormalignant cell. In addition, as known, the formation of a benign ormalignant tumor is dependent, inter alia, on continuous divisions of thecells forming the tumor. Administration of the dorman compositions to anindividual before the formation or at early stages of the formation of abenign or malignant tumor may result in the delay or prevention of theformation of a fully fledged tumor in the treated individual.Administration of said extracts to an individual suffering from a fullyfledged benign or malignant tumor may result in the reduction of thetumor load in the treated individual and in the alleviation of thetumor-related symptoms. Said dorman compositions may be effective in thetreatment of primary as well as secondary (metastatic) tumors. Saidextracts may also be administered in combination with one or more knownanti-tumorigenic treatments (e.g. chemotherapeutic agents, radiationetc.) to achieve a synergistic anti-tumorigenic effect. The doses ofsaid extracts to be administered to an individual as well as thetreatment modality will be dependent on characteristics of the treatedindividual (age, weight, medical history, etc.) as well as oncharacteristics of the developing or existing tumor (benign ormalignant, size, origin, primary or secondary, etc.). In individualshaving a high risk of developing a primary or secondary tumor, thedorman compositions may be administered routinely in order to reduce theprobability of tumor formation.

The present invention thus further provides a composition comprising adorman extract capable of inhibiting the proliferation of cells, for theadministration to an individual having a benign or malignant tumor orbeing at a high risk of developing a tumor.

By yet an additional aspect of the invention, dorman compositions may beused to enhance the therapeutic index of chemotherapeutic and radiationtreatments. In an individual receiving such treatments, normal dividingcells such as cells of the inner lining of the intestines, cells of hairfollicles and hematopoietic cells are also harmed by thechemotherapeutic agents or radiation which are aimed at destroying themalignant cells of which a large percent are dividing cells. Byadministration of dorman compositions to an individual prior to ortogether with such treatments, it may be possible to inhibit theproliferation of a significant percent of the normal cells. As a result,toxic side effects due to the influence of the treatments on normalcells may be significantly reduced and when beneficial, higherconcentrations of the chemotherapeutic or radiation treatments may beused. In order to facilitate the toxicity reducing affect of the dormancomposition, it may at times be administered directly to a needing site,tissue or organ, e.g. onto the skin.

The present invention thus provides by a further of its aspects, acomposition capable of inhibiting the proliferation of cells, foradministration to an individual receiving chemotherapeutic or radiationtreatments, comprising an effective amount of dorman, dorman extract ordorman analog.

Other therapeutic applications of the dorman compositions includeinhibition of fibrosis, e.g. skin fibrosis, cirrhosis, and others. Itshould be noted that hitherto, fibrosis, which is an over proliferationof fibroblasts, has been treated by cytotoxic drugs, but with a limitedapplication due to their general non specific toxicity. Inhibition ofthe fibroblast proliferation by the use of the dorman composition of theinvention, provides a viable, less toxic alternative. In a similarmanner, the dorman compositions of the invention may also be useful inthe treatment of psoriasis which results from over proliferation ofkeratinocytes. Seborrheic keratosis, papilomas and warts may also betreated by the dorman compositions.

Another possible application of the dorman composition of the inventionis in preservation of organs or tissue prior to their use fortransplantation.

Other applications of said dorman compositions may be, for example, inthe treatment of scalp baldness (Alopecia) which is many times one ofthe phenomenas associated with aging of the skin in an individual. Inindividuals suffering from Alopecia, the life span of scalp hairdecreases substantially (e.g. from a life span of about 3 years in anormal individual to a life span of about one year in an individualsuffering from Alopecia). Therefore, decreasing the rate of hair growthin an individual having a high probability of developing Alopecia or inan individual already showing for signs of scalp hair loss, willdecrease the extent of such hair loss. Administration of the dormancompositions of the invention to such an individual may result in apartial or complete decrease of the hair loss. For this purpose, thedorman comprising compositions may be administered either topically atthe site of scalp hair loss or, alternatively, in other cases may beadministered systemically.

An additional phenomena which may be treated by administration of thedorman comprising compositions of the invention is associated withovergrowth of hair in various parts of an individual's body, such asarms, back, etc. (Hirsutism). Such undesired overgrowth of hair appearsmany times in aging individuals and, at times, is associated with lossof scalp hair in the same individual. Due to their ability to reducecell growth, compositions of the invention may be useful in reducingsuch undesired overgrowth of hair.

The amount of the dorman comprising compositions to be administered forthe above two indications, the administration regimes as well as theirmode of application will again depend both on characteristics of thetreated individual (age, size, gender, etc.) as well as on parametersassociated with the phenomena to be treated (such as the extent of scalphair loss, the specific body parts in which there is overgrowth of hair,etc.).

In addition, the dorman composition may be useful as a complementaryagent administered in combination with or following hair removaltreatments such as, for example, shaving (where said extract may beincorporated in an aftershave solution) or hair stripping (e.g. by wax).

Another application of the dorman composition may involve itsadministration to an individual during the period in which a scar isformed, e.g. after an operation in order to decrease scar formation. Byslowing down the rate of the healing process in such an individual, thefinal scar may be much less apparent. In addition, the anti-fibroticeffect of the dorman compositions decreases the formation of cheloidswhich commonly appear after healing.

The dorman composition may also be useful for extending the duration ofa tan in an individual. Following exposure to the sun, epidermal cellscomprise a high concentration of melanin. During skin renewal suchmelanin comprising cells are shed. By slowing down the cell renewalprocess in the skin, the dorman composition causes the melanincomprising cells and thus the tan to remain for a longer period of time.

In addition to inhibiting proliferation of various cells, said dormancompositions are also capable of slowing seed germination and inhibitinggrowth of various plant seedlings. Following germination of seeds, rootsand hypocotyls begin to develop in the seedling. Incubation of the plantseedlings with the dorman composition results in the inhibition of theelongation of the seedling roots and hypocotyls. The dorman compositionsmay therefore be used for weed control, wherein their administration atan appropriate concentration may result in the inhibition of growth ofnon desirable weeds while not affecting the growth of the desired plant.In view of the natural origin of the dormans, their administration hasno noticeable toxic effect on cells or tissue on which they are inducedto act or on the environment. In addition, at times it may be useful touse such compositions for long term storage of seeds and seedlings.

The present invention thus provides a dorman composition having theactivity of slowing and inhibiting the growth of plant seeds and/orseedlings comprising said dorman extract.

A further application of the dorman composition of the invention is inthe preservation of fresh produce, e.g. vegetables, fresh fish eggs,fish shells, etc.

The invention also provides a process for the preparation of ananti-proliferative composition comprising mixing dormans or DC with acarrier so as to yield an anti-proliferative composition with ananti-proliferative effective amount of dormans in said composition. Sucha prepared composition may be used, depending on the nature of thecarrier, in therapy, cosmetics, food preservation or agriculture. Such aprocess for preparing a pharmaceutical or cosmetic composition typicallycomprises preparing an DC, and mixing it with an appropriatepharmaceutical or cosmetic acceptable carrier, the amount of DC beingsuch so as to yield a final therapeutically or cosmetically (as the casemay be) effective amount of dormans in the composition. Also provided isuse of dormans or an DC for the preparation of such a pharmaceutical orcosmetic composition.

As will be appreciated, the various applications of the dormancomposition of the invention given above, are examples of a myriad ofpossible applications of these compositions, all having in common theinhibition of proliferation of target cells.

In the following, the invention will be illustrated by some non-limitingexamples with occasional reference to the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a histogram showing the number of keratinocytes in a cellculture well at different periods of time after their incubation withvarious concentrations of the DC IBR-1 (obtained from a dormant plant(FIG. 1A) or the AE IBR-3 (obtained from an active plant) (FIG. 1B). Asa control, the cells were incubated with growth medium alone (0 μg/ml ofthe tested DC or AE). The number of cells in each well was determined bythe microculture methylene blue assay (described in Example II) and thedyed culture plates were read at 620 nm.

FIG. 2 is a histogram showing the number of fibroblasts in amicroculture at different periods of time after their incubation withvarious concentrations of the following

FIG. 2A—cells were incubated with the DC IBR-1 (dormant);

FIG. 2B—cells were incubated with the AE 'BR-3 (active).

FIG. 3 is a histogram showing the number of keratinocytes in amicroculture at different periods of time after their incubation withvarious concentrations of the cell culture derived DC IBR-11. Ascontrol, the cells were incubated with growth medium only (0 μg/ml DEIBR-11). The number of cells in each well was determined by themicroculture methylene blue assay (described in Example II) and the dyedculture plates were read as 620 mn.

FIG. 4 is a histogram showing the number of fibroblasts in amicroculture at different periods of time after their incubation withvarious concentrations of the cell culture derived DC IBR-11.

FIG. 5 is a graphic representation showing the number of cultured mousebladder carcinoma cells (T24P) in a microculture 72 hours after theirincubation with various concentrations of a plant derived DC (IBR-1)(FIG. 5A) or an animal derived DC (IBR-4) (FIG. 5B). The number of cellsin each well was determined by the microculture methylene blue assay(described in Example 2) and the dyed culture plates were read at 620nm.

FIG. 6 is a graphic presentation showing the number of mouse bladdercarcinoma cells (T50) in a microculture at different periods of timeafter their incubation with various concentrations of DC IBR-1. Thedorman extracts were added at day 1 and day 3 of the cell culture andthe number of cells in each well were determined on day 5 of the cultureas described in FIG. 5 above. The number of cells in each tested wellwas determined by using the microculture methylene blue assay asexplained in the description of FIG. 1 above and by reading of the dyedmicroculture plate at 620 nm.

FIG. 7 is a histogram showing the DNA content analysis of keratinocytesincubated with DC EBR-1 for 2 and 5 days (7A and B, respectively) AEIBR-3 (7C and D, respectively) and DC IBR-4 (7E and F, respectively).The analysis was carried out with a FACS, FPAR-Plus (Becton-Dickinson,Inc.) using ethidium bromide. The percent of cells being in the G1phase, S phase and G₂+M phase was determined in each cell culture. Inaddition, the percent of apoptosis (A) in each culture was alsodetermined.

FIG. 8 is a histogram showing the DNA content analysis of fibroblastincubated with DC IBR-1 for 2 and 5 days (FIGS. 8A and B), AE IBR-3(FIGS. 8C and D) and DC IBR-4 (FIGS. 8E and F). The analysis was carriedout as described in FIG. 7 above.

FIG. 9 is a graphic representation showing the UV spectra of bandsobtained by separating by thin layer chromatography (TLC).

FIG. 10 is a graphic representation showing the comparative effect ofdifferent treatments on the duration of a tan 5 days and 17 days afteradministration of the cream on to the tanned area:

FIG. 10A shows the effect of a cream containing 5% IBR-1 on theelongation of the duration of a tan 5 days after administration of thecreams.

FIG. 10B shows the same effect as FIG. 10A 17 days after administrationof the creams.

FIG. 10C shows the effect on the duration of the tan (shortening) of acream comprising alpha hydroxy acid (AHA) as compared to the tan withoutcream 5 days after administration of the creams.

FIG. 10D shows the same effect as FIG. 10C 17 days after administrationof the cream.

EXAMPLES Example I A: Effect of Narcissus Bulb DC Prepared fromNarcissus Field Bulbs on Growth of Cucumber Seedlings

(a) Inducing dormancy in Narcissus field bulbs

Narcissus field bulbs were obtained and subjected to hot water having atemperature of 45° C. for 2-4 hours. The bulbs were then either usedimmediately for the preparation of water soluble extracts or,alternatively, maintained in a dry room at a temperature of 30° C. for amaximal period of 8 months after which they were used for thepreparation of the plant extract.

(b) Preparation of extracts from Narcissus bulbs

Active or dormant Narcissus field bulbs (induced into dormancy asexplained above) were disinfected in soap water for a period of 1 hour.The bulbs were then cut and homogenized in distilled water (30 sec×3)using a Homogenizer Ultra-Turbo-turax. The homogenized preparation ofthe bulbs was then filtrated through a first 0.45 m sterile filter andthen through a second 0.22 μm filter and the preparation which was notmaintained on the filters was then collected. The concentration isdefined as weight of original bulb (gr.) per final extract volume (ml).

(c) The effect of the Narcissus field bulb extracts on growth ofcucumber seedlings

(i) Experimental Assay

Cucumber seeds cv. “Delila”, 1994-1995, 95% germination, 99.9% puritywere germinated in tap water and then incubated in the dark at 27° C.for about 20 hours until root initiation (1-2 mm). Each experimentalgroup comprised Petri dishes which were filled each with 2 mis of DC(IBR-1) originated from dormant Narcissus field bulbs obtained asexplained above in various concentrations.

One layer of filter paper was placed in each of the petri dishes and 10cucumber seeds, germinated as explained above, were placed on the paper.The petri dishes were incubated for 24 to 72 hours at 26° C. to 28° C.in the dark.

The effect of the tested bulb extracts in various concentrations weretested on two parameters of the cucumber seeds:

1. Root elongation

2. Hypocotyl elongation.

These two parameters were tested every 24 hours after the incubation ofthe seeds with the tested extract and every 24 hours after that.

(ii) Results

As seen in Table 3 below, DC IBR-1 showed sufficient inhibitive activityon the seedlings'growth, as measured by the length of the roots andhypocotyls of seeds which were incubated with DC IBR-1 compared to thelength of the same organs incubated with sterile water. The inhibitiveactivity of DC IBR-1 was dose dependent.

TABLE 3 Growth of seeds with and without extract: 24 hrs 48 hrs RootHypocotyl Root Hypocotyl Extract source (mm) (mm) (mm) (mm) SterileWater 30 2 47 7 DC from Narcissus 0.2 gr./ml  4 0  4 1 0.1 gr./ml  5 0 5 2 0.05 gr./ml  6 0.5 10 2 0.01 gr./ml 16 1 26 4 0.005 gr./ml 22 2 357 0.001 gr./ml 25 2 41 7 Inhibition (%) of seed growth by extracts: 24hrs 48 hrs (% inhibition) (% inhibition) Extract source Root HypocotylRoot Hypocotyl Sterile Water  0 0  0  0 DC from Narcissus 0.2 gr./ml 87100 91 86 0.1 gr./ml 83 100 89 71 0.05 gr./ml 80 75 79 71 0.01 gr./ml 4750 45 43 0.005 gr./ml 27 0 25  0 0.001 gr./ml 17 0 13  0

Example I B: Reversibility of the Effect of Narcissus DC on Growth ofCucumber Seedlings (i) Experimental Assay

(a) The experiment was conducted in an identical manner to thatdescribed in I(A) above. The effect of the DC on growth of roots andhypocotyls of cucumber seeds was measured 24 and 72 hours afterincubation. 72 hours after incubation, the seeds were washed withsterile distilled water and incubated with sterile distilled water foran additional 72 hours at 27° C. in the dark. The length of the rootsand hypocotyls of the seeds was measured again 144 hours after thebeginning of incubation (72 hours after washing away the DC).

(ii) Results

As seen in Table 4 below, after washing away the Narcissus derived DCwhich had an inhibitive effect on the growth of roots and hypocotyls ofcucumber seeds, the roots and hypocotyls began to grow again. Thus, theinhibitive effect of DC was reversible and non toxic. The same effectwas apparent at lower concentrations of the DC incubated with the seedsas well (results not shown).

TABLE 4 144 H (72 Hours 24 H 72 H after washing) Root Hyptocotyl RootHyptoctyl Root Hypocotyl Time Extract (mm) (mm) (mm) (mm) (mm) (mm)Sterile water 44.9 6.5 111.7 17.5 — — DC 0.2 gr/ml 2.6 1.5 2.7 6.2 43.322.9

Example II Effect of Narcissus Bulb Derived DC on Proliferation ofKeratinocytes

(a) Preparation of keratinocyte cultures

Human keratinocyte cultures were prepared as described in Ben Bassat H.,et al., Plastic and Reconstructive Surgery, 89:511, (1992). Generallykeratinocyte cultures were initiated from small biopsy specimens (about1 cm²) of split-thickness skin. The biopsy specimens from healthy donorswere obtained under local anesthesia with 1% lidocaine. The biopsy wasincubated in trypsin-EDTA at 4° C. for 18-20 hours. Thereafter, theepidermis was separated and the epithelium desegregated in trypsin-EDTAto form a single cell suspension. Trypsin 0.125%-EDTA 0.025% in Puck'ssaline with ×10 antibiotics, 1000 U/ml penicillin, 1000 μg/mlstreptomycin, 0.0025 μg/ml amphotericin B and 0.4 mg/ml gentarnycin wereused for these procedures. Trypsin solutions were prepared from trypsin1:250 strength.

The cell suspensions prepared as described above, were inoculated at aconcentration of 3-6×10⁶ cells into 25 cm² Falcon flasks which werepre-prepared to contain 2×10⁵ lethally irradiated 3T3 mouse fibroblastsas a feeder layer.

The flasks were incubated at 37° C. 10% CO₂ for about 8-10 days untilthe cultures were about 80% confluent. At this stage, the cells in eachflask were released by addition of trypsin 0.25% —EDTA 0.05% (1:1)without antibiotics and the released cells after being washed wereinoculated into 96-well microplate at a concentration of 3×10⁴ cells perwell without feeder layers in keratinocyte medium (Kmed) according toRheinwald and Green (Rheinwald T. G. and Green, H., Nature, 265:421-424(1988)) to form a secondary culture.

(b) Effect of Narcissus bulb derived DC on the proliferation ofkeratinocytes

(i) Experimental Assay

Narcissus bulb extracts were obtained as described in Example I(b) abovefrom active and dormant bulbs. The secondary keratinocyte cell culturesseeded in microplates as described above, were further grown in Kmed fora period of 3-4 days. The microplates were then divided into thefollowing main groups:

(1) keratinocytes which were continuously grown in Kmed;

(2) keratinocytes grown in Kmed containing the AE IBR-3 prepared fromactive Narcissus bulbs in several concentrations (from 0.5 μg/ml-10μg/ml, each concentration forming a separate experimental group) and

(3) keratinocytes grown in Kmed medium comprising the DC IBR-1 obtainedfrom dormant Narcissus bulbs at various concentrations (0.5 μg/ml-10μg/ml each concentration forming a separate experimental group).

Each experimental group contained 5 wells. The growth medium containingDC IBR-1 or AE IBR-3 was changed every 24 hours for a period of 5 daysafter which the medium was removed from all of the wells and fresh Kmedmedium without any plant extract was added and the cultures were grownin it for an additional 3 days.

The effect of the tested DC or AE on the proliferation of thekeratinocytes was determined by the number of cells detected in a testedtreated well as compared to the number of cells detected in a well inwhich the cells grew in Kmed medium without any DC or AE.

The number of cells in each well was determined by the microculturemethylene blue assay as follows:

Extract treated cultures and controls were fixed in glutaraldehyde,0.05% final concentration, for 10 mins. at room temperature. Afterwashing, the microplates were stained with methylene blue 1% in 0.1 Mborate buffer pH 8.5 for 60 mins. at room temperature. Thereafter theplates were extensively and rigorously washed to remove excess dye anddried. The dye taken up by cells is eluted in 0.1 N HCl for 60 mins. at37° C. and read at 620 nm.

In preliminary titration experiments linear readings were obtained for1×10³ to 4×10⁴ cells/well. Each point of the growth curve experiments isan average of the reading of 5 wells, since keratinocytes grow inislands and do not form uniform monolayers. The number of average cellsin the wells was determined at 2 days, and 5 days after incubation ofthe keratinocytes with the tested DC or AE as well as at 8 days afterincubation (following 3 days growth without the tested extract).

(ii) Results

As can be seen in FIG. 1A, DC IBR-1 from Narcissus bulbs had asignificant inhibitory effect on the proliferation of keratinocytes. Theinhibition was apparent from day 5 of the experiment and was dosedependent. Inhibition of the keratinocyte proliferation was apparent ata concentration as low as 0.5 μg/ml but was most significant at aconcentration of 10 μg/ml of the DC. The effect was dose dependent(starting at a concentration of 1 g/ml of the DC and most effective at aconcentration of 10 μg/ml of the DC).

Against this, as seen in FIG. 1B, AE IBR-3 showed no significantinhibitory effect on the proliferation of keratinocytes.

Example III Effect of DC Obtained from Dormant and Active NarcissusBulbs on the Proliferation of Fibroblast in Culture

(a) Preparation of fibroblast cell cultures:

Primary fibroblast cell cultures were initiated from small human skinspecimens and prepared as described in Example II above regardingpreparation of keratinocyte cultures except that the growth medium usedwas DMEM+20% fetal calf serum.

(b) Preparation of DC and AE

DC IBR-1 and AE IBR-3 were prepared from dormant and active Narcissusbulbs as described above.

(c) Effect of DC and AE on fibroblast proliferation:

(i) Experimental Assay:

The above extracts were added to the fibroblast cultures at variousconcentrations (1 g-10 g/ml) and the number of fibroblasts in thecultures was determined 2 days, 5 days and 8 days after the addition ofthe extracts to the cells as described in Example II (a) above.

(ii) Results

As seen in FIG. 2A, DC IBR-1 had the most significant inhibitory effecton the proliferation of fibroblast in culture. The effect was apparentfrom day 5 of the experiment and although was dose dependent, the effectwas seen at doses as low as 0.5 μg/ml.

As can be seen in FIG. 2B, AE IBR-3 had no inhibitory effect on theproliferation of fibroblasts.

Example IV Preparation of Cosmetic and Dermatological CompositionsComprising DC

The following are several specific examples of cosmetic anddermatological compositions which may be used in accordance with theinvention for administration to an individual.

A. Balm (topical route): • Ozokerite 10 gr. • Isopropyl palmitate 9 gr.• White vaseline 14 gr. • Preserving agent 0.2 gr. • Antioxidants 0.3gr. • Perfume 1 gr. • DC prepared from bulb extract 0.00001 gr. • Liquidparaffin qs 100 gr. B. Balm (topical route): • Ozokerite 19 gr. • Liquidpurcellin oil 10 gr. • White vaseline 15 gr. • Preserving agent 0.2 gr.• Antioxidant 0.3 gr. • DC prepared from bulb extract 0.00002 gr. •Liquid paraffin qs 100 gr. C. Emulsified gel of O/W type (topicalroute): • Carbopol ® 981 (marketed by Goodrich) 0.6 gr. • Volatilesilicone oil 3 gr. • Purcellin oil 7 gr. • Preserving agent 0.3 gr. •Ethyl alcohol 15 gr. • Perfume 0.4 gr. • Triethanolamine 0.2 gr. • DCprepared from bulb extract 0.04 gr. • Demineralized water qs 100 gr. D.Aqueous-alcoholic gel (topical route): • Carbopol ® 981 (marketed byGoodrich) 1 gr. • Triethanolamine 1 gr. • 95% Ethanol 60 gr. • Glycerol3 gr. • Propylene glycol 2 gr. • DC prepared from bulb extract 5 gr. •Demineralized water qs 100 gr. E. Anhydrous gel (topical route): •Absolute ethanol 61,1992 gr. • Hydroxyethyl cellulose 0.8 gr. •Propylene glycol 25 gr. • Polyethylene glycol 12 gr. • DC prepared frombulb extract 0.0008 g F. Emulsion of O/W type (topical route): •Volatile silicone oil 10 gr. • Liquid paraffin 6 gr. • Liquid lanolin 3gr. • Arlacel ® 165 (marketed by Atlas) 6 gr. • Tween ® 60 (marketed byAtlas) 2 gr. • Cetyl alcohol 1.2 gr. • Stearic acid 2.5 gr. •Triethanolamine 0.1 gr. • Preserving agent 0.3 gr. • Antioxidants 0.3gr. • DC prepared from bulb extract 0.5 gr. • Demineralized water qs 100gr. G. Emulsion of O/W type (topical route): • Propylene glycol 2 gr. •PEG 400 3 gr. • Preserving agent 0.3 gr. • Carbopol ® 981 (marketed byGoodrich) 0.2 gr. • Isopropyl myristate 1 gr. • Cetyl alcohol 3 gr. •Stearic acid 3 gr. • Glycerol 3 gr. • Corn oil 2 gr. • Perfume 0.5 gr. •DC prepared from bulb extract 0.001 gr. • Demineralized water qs 100 gr.H. Clear gel (topical route) • Oxyethylenated nonylphenol 5 gr. •Carbopol ® 981 (marketed by Goodrich) 1 gr. • Ethyl alcohol 30 gr. •Triethanolamine 0.3 gr. • Glycerine 3 gr. • Perfume 0.3 gr. • Preservingagent 0.3 gr. • DC prepared from bulb extract 1 gr. • Demineralizedwater qs 100 gr. I. Cream containing liposomes (topical route): • Cetylalcohol 4 gr. • B-sitosterol 4 gr. • Dicetyl phosphate 0.5 gr. •Preserving agent 0.3 gr. • Sunflower oil 35 gr. • Perfume 0.6 gr. •Carbopol ® 981 (marketed by Goodrich) 0.2 gr. • Triethanolamine 0.2 gr.• Sphingosine 0.05 gr. • DC prepared from bulb extract 0.2 gr. •Demineralized water qs 100 gr. J. Per os composition: • Talc 5 mg •Aerosil 200 5 mg • Stearate de Zn 5 mg • DC prepared from bulb extract 3mg • Lactose qs 400 mg K. Liquid for Iontophoresis: • Benzoate de sodium2 mg • Preserving agent 0.15 gr. • DC prepared from bulb extract 1 gr. •Water qs 100 gr. L. Emulsion W/O: • Protegin (marketed by Goldschmidt)19 gr. • Vaseline oil 8 gr. • Glycerine 3 gr. • DC prepared from bulbextract 1 gr. • Sulfate de Mg 0.5 gr. • Perfume 0.8 gr. • Preservingagent 0.2 gr. • Water qs 100 gr.

Example V Extracts prepared from Narcissus Bulblets (cell cultures) onCucumber Seedling Growth:

(a) Preparation of Narcissus bulb cell cultures

Active Narcissus bulbs from the field having inflorescence stalkinitials were used to prepare duplicate inner scale explants. Theexplants were then inoculated into NR31 medium (NAA/10 μM BA: 5:0.5 μM)to initiate callus cultures. Four to 5 weeks after the initiation of thecallus cultures, the cultures were transplanted into NRS medium (6%sucrose) to form bulblets growth explants. For scaling up of thebiomass, half bulblets were transplanted into bulblet column bioreactorswith liquid basal media N4 comprising

Murashige & Skoog (Sigma M-5525) 4.33 gr/L Myoinositol 100 mg/L Adeninesulfate 150 mg/L NaH₂PO₄H₂O 345 mg/L NAA 5 μM Agar Type A 7 gr/L BA 5 μMpH = 5.7 Pyridoxine 1 mg/L Glycine 2 mg/L Nicotinic acid 5 mg/L ThiamineHCl 0.5 mg/L Sucrose 30 gr/L

for a period of 4 weeks.

(b) Preparation of cell culture derived DC

The plant material prepared as described in (a) above was then shakenfor 7-10 days on a gyratory shaker at about 35° C. (the column weightper medium was 0.1 gr/ml). Half of the cell cultures were induced intodormancy by their incubation at a high temperature (of about 35° C.).The DC prepared from such cultures was designated IBR-11. The remainingcell cultures were maintained in their active state by growing them inregular conditions and the AE prepared from them was designated EBR- 10.AE IBR-10 or DC IBR-11 were prepared from the medium free bioimass whichwas washed with water, weighed and homogenized in an ultra-Turbo-turax.The homogenate was suspended and diluted in steril distilled water.

(c) Cucumber seeds cv. “Delila”, 1994-1995, 95% germination, 99.9%purity were germinated in tap water at 27° C. in the dark for about 20hours until root initiation (1-2 mm).

(d) The experimental Assay

(i) The effect of the above DC and AE prepared from Narcissus bulbletson root elongation and hypocotyl elongation of the cucumber seedlingswas determined as follows. Each experimental group consisted of petridishes each containing 10 seeds was incubated with:

1. DC IBR-11 (dormant)

2. AE IBR-10 (active).

One or two layers of filter paper were placed in each of the petridishes and 10 cucumber seeds, terminated as explained above, were placedon the filters. The Petri dishes were incubated for 72 hours at 27° C.to 30° C. in the dark.

The effect of extracts was tested on two parameters of the cucumberseeds:

1. Root elongation

2. Hypocotyl elongation.

These two parameters were tested after 72 hours of incubation of theseeds with the extract.

(ii) Results

As seen in Table 5 below, 72 hours after incubation, DC IBR-11 had asignificantly higher inhibition activity on both the root length andhypocotyl length of the seedlings as compared to the effect of AE on theseedlings' growth.

TABLE 5 % inhibition of DC compared to AE AE (IBR-10) DC (IBR-11)$\frac{\left. {({AE}) - {DC}} \right)}{({AE})} \times 100$

Root length (mm) 72 h 86 31 58% Hypocotyl (mm) 72 h 81 49 40%

Example VI Effect of Narcissus Bulb Cell Culture Derived DC onProliferation of Keratinocytes

(a) Preparation of keratinocyte cultures

Human keratinocyte cultures were prepared as described in Example II (a)above.

(b) Effect of Narcissus bulblet derived DC on the proliferation ofkeratinocytes

(i) Experimental Assay

Narcissus bulb derived cell cultures were obtained as described aboveand DC IBR-11 was prepared from bulblets induced into dormancy (as isalso described above).

The secondary keratinocyte cell cultures seeded in microplates asdescribed above, were further grown in Kmed for a period of 3-4 days.The microplates were then divided into the following main groups:

(1) keratinocytes which were continuously grown in Kmed; and

(2) keratinocytes grown in Kmed containing DC IBR-1 in severalconcentrations (from 0.5 μg/ml-10 μg/ml, each concentration forming aseparate experimental group).

Each experimental group contained 5 wells. The growth medium containingDC was changed every 24 hours for a period of 5 days after which themedium was removed from all of the wells and fresh Kmed medium withoutany plant extract was added and the cultures were grown in it for anadditional 3 days.

The effect of the tested DC on the proliferation of the keratinocyteswas determined by the number of cells detected in a tested treated wellas compared to the number of cells detected in a well in which the cellsgrew in Kmed medium without any DC.

The number of cells in each well was determined by the microculturemethylene blue assay as follows:

Extract treated cultures and controls were fixed in glutaraldehyde,0.05% final concentration, for 10 mins. at room temperature. Afterwashing, the microplates were stained with methylene blue 1% in 0.1 Mborate buffer pH 8.5 for 60 mins. at room temperature. Thereafter theplates were extensively and rigorously washed to remove excess dye anddried. The dye taken up by cells is eluted in 0.1 N HCl for 60 mins. at37° C., and read at 620 nm.

In preliminary titration experiments linear readings were obtained for1×10³ to 4×10⁴ cells/well. Each point of the growth curve experiments isan average of the reading of 5 wells, since keratinocytes grow inislands and do not form uniform monolayers. The number of average cellsin the wells was determined at 2 days, and 5 days after incubation ofthe keratinocytes with the tested DC as well as at 8 days afterincubation (following 3 days growth without the tested extract).

(ii) Results

As can be seen in FIG. 3, DC IBR-11 showed significant inhibitoryactivity on the proliferation of keratinocytes in culture.

Example VII Effect of DC Obtained from Dormant Narcissus bulblets on theProliferation of Fibroblast in Culture

(a) Preparation of fibroblast cell cultures:

Primary fibroblast cell cultures were initiated from small human skinspecimens and prepared as described in Example II above regardingpreparation of keratinocyte cultures except that the growth medium usedwas DMEM+20% fetal calf serum.

(b) Preparation of cell cultured DC:

IBR-11 was prepared from dormant bulblets as described in Example V(b)above.

(c) Effect of DC on fibroblast proliferation:

(i) Experimental Assay

The above extracts were added to the fibroblast cultures at variousconcentrations (1 μg-10 μg/ml) and the number of fibroblasts in thecultures was determined 2 days, 5 days and 8 days after the addition ofthe extracts to the cells as described in Example V(a) above.

(ii) Results

As can be seen in FIG. 4, the cell culture derived DC IBR-11 showedinhibitory activity on the proliferation of fibroblasts.

Example VIII Effect of Fruit Juice on Cucumber Seed Growth

(a) Preparation of fruit juice

Grapefruit juice was produced from one fresh grapefruit and the producedjuice was squeezed and filtrated through cotton cloth and thencentrifuged at 10.000 rpm for 10 mins. at room temperature. Thesupenatant was then used for testing its effect on cucumber seed growthas described below.

(b) The effect of grapefruit juice on growth of cucumber seedlings:

(i) Experimental Assay

Cucumber seeds were prepared as described in Example 1 (c)(i) above.Each experimental group comprised ten Petri dishes which were filledwith 1.8 ml of the following:

(1) dH₂O

(2) Fruit juice obtained as in (a) above.

One or two layers of filter paper were placed in each of the petridishes and 10 cucumber seeds, germinated as explained above, were placedon the filters. The petri dishes were incubated at 25° C. and theparameters of the cucumber seeds were measured at 24 hours and 72 hoursafter bezinring of incubation.

The effect of the fruit juice was tested on two parameters of thecucumber seeds:

(1) Root elongation

(2) Hypocotyl elongation

(ii) Results

As seen in Table 6 below, this experiment indicates that the fruit juicecomprises inhibitory activity on the growth of cucumber seeds.

TABLE 6 72 hours 72 hours % inhibition Treatment Root mm Hypocotyl mmRoot mm Hypocotyl mm dH₂O 110 35 — — Grapefruit  3  0 97 100 Juice

Example IX Effect of dorman Extract Obtained from the Crustacean Artemiasalina on Cucumber Seed Growth

(a) Preparation of extracts from Artemia salina:

The dorman extract designated IBR-4 was obtained by preparing an extractfrom Artemia salina. The Artemia “eggs ” may be submitted to dehydrationor high salt concentration resulting in opening of their shell which isthen followed by their grounding. Alternatively, the Artemia eggs may bedispensed in 10 ml of water resulting in softening of the shell.Following grounding or softening of the shells, the eggs are thendissolved in one of any of solvents known per se (e.g. water) to obtainan extract from them. The extract may be lyophilized (as in thisexample) or alternatively, used as obtained. An additional method ofobtaining the Artemia extract may be to dissolve the Artemia eggs inwater until the prenauplius larvae crawl out of the shells after whichthe larvae are grounded and an extract obtained therefrom.

(b) The effect of the DC extract from Artemia (IBR-4) on Growth ofcucumber seedlings:

(i) Experimental Assay

Cucumber seeds were prepared and seeded into petri dishes as explainedin Example 1 above. Each experimental group comprised a petri dishcontaining 10 cucumber seeds and the results shown below are an averageof the parameters measured for the 10 seeds. The petri dishes were grownat 28° C. in the dark and the root length and hypocotyl length of thecucumber seeds were measured 24 hours and 48 hours after beginning ofincubation with 1.8 ml of one of the following:

(1) dH₂O

(2) DC IBR4 at a concentration of 0.02 gr./ml.

(ii) Results

As seen in Table 7 below, the Artemia dorman extract IBR-4 had asignificant inhibitory effect on cucumber seed growth which was apparentalready after 24 hours of incubation of the seeds with the DC IBR4 butwas most significant 48 hours after incubation (66% inhibition on rootgrowth and 40% inhibition on hypocotyl growth).

TABLE 7 24 hours 24 hours 48 hours 48 hours Root Hypocotyl % InhibitionRoot Hypocotyl % inhibition Treatment mm mm Root Hypocotyl mm mm RootHypocotyl dH₂O 35 4  0  0 61 10  0  0 IBR4 0.02 gr/ml 20 3 43 25 21  666 40

Example X Effect of a Dorman Extract from a Dormant Plant (Narcissusderived IBR-1) and a Dorman Extract Obtained from Animals (Artemiaderived IBR-4) on Proliferation of Mouse Bladder Carcinoma Cells

(a) Preparation of the dorman extracts:

The Narcissus derived plant dorman extract IBR-1 and the Artemia derivedanimal dorman extract IBR-4 were prepared as explained in the Examplesabove.

(b) The effect of DC IBR-1 and DC IBR-4 on proliferation of mousebladder carcinoma cells:

(i) Experimental Assay

The two experiments were carried out as follows:

T24P cells (mouse bladder carcinoma cells) (FIG. 5) were plated at aconcentration of 5×10⁴ cells/well in a microculture well and T50 cells(mouse bladder carcinoma cells) (FIG. 6) were plated at a concentrationof 2×10⁴ cells per well in a microculture plate and the cells were grownin cell culture medium.

DC IBR-1 and DC IBR4 were added to the T24P cell cultures and IBR-1 wasadded also to the T50 cells at various concentrations (0 g/ml-25 g/ml)24 hours after plating of the cells and 48 hours after the beginning ofincubation of the cells with the dorman extracts. The number of cellsper well were determined 48 hours after the beginning of incubation ofthe cells with the extracts using the microculture methylene blue assay(described in Example 2 above) and the dyed culture plates were read at620 nm.

(ii) Results

As seen in FIGS. 5 and 6, both the plant derived dorman extract as wellas the animal derived dorman extract had some inhibitory effect on theproliferation of mouse bladder carcinoma cells T24P (FIG. 5) and T50(FIG. 6).

Example XI Inhibition of Cucumber Seed Growth by Grape and Kiwi Juice

(a) Preparation of fruit juice

Grape and Kiwi juices were produced from fresh fruit by blending thefruit in the blender cup for 3 mins. at high speed, filtrating the blendthrough a cheese cloth and centrifuging it at 6,500 rpm for 10 mins. atroom temperature. The supernatant was then used for the experiment at aconcentration of 1.2 gr/ml of the grape juice and 1.26 gr/ml of the kiwijuice. The concentration was determined by fruit original weight (gr.)for final volume (v). Several dilutions of each juice were prepared andused for testing on cucumber seed growth.

Preparation of the cucumber seeds and the experimental assay werecarried out as described in Example l(c)(i) above. The length of theroots and hypocotyls were measured 24 and 48 hours after beginning ofincubation of the seeds with each of the tested juices or controls at28° C. As seen in Table 8 below, both the kiwi juice and the grape juiceshowed a very high percent of inhibition both on growth of cucumberroots as well as on hypocotyls. The most prominent inhibition was seen48 hours after beginning of incubation wherein both juices inhibited thegrowth of the cucumber seeds.

TABLE 8 24 h 48 h Root % Hypocotyl % Root % Hypocotyl % InhibitionInhibition Inhibition Inhibition dH₂O 0 0 0 0 Kiwi juice 50 100 72 100100 mg/ml Kiwi juice 50 mg/ml 33 67 55 63 Kiwi juice 25 mg/ml 0 67 42 38Kiwi juice 5 mg/ml 0 0 7 13 Grape juice 83 100 88 100 100 mg/ml Grapejuice 40 100 67 63 50 mg/ml Grape juice 17 67 42 63 25 mg/ml Grape juice5 mg/ml 10 17 20 25

Example XII Cell Cycle Analysis of Cells After Incubation with DC (i)Experimental Assay

Cell cultures of keratinocytes obtained from healthy human adults andcell cultures of fibroblasts obtained from healthy human skinpreparations were incubated with Narcissus derived DC (IBR-1), Narcissusderived AE (IBR-3) and Artemia derived DC (EBR-4) at variousconcentrations. The DNA content of the cells was analyzed by FACS usingethidium iodide as the fluorescent dye which binds to the DNA (Parks D.R., and Herzenberg, L. A., In: Methods in Cell Biology, Vol. 26,Academic Press, p. 283, 1982). The analysis was carried out on day 2 and5 after beginning of incubation of the cells with the various extractsand was carried out with FACS FPAR-Plus (Becton-Dickinson, Inc.)

In addition, the percent apoptosis in each cell culture incubated withthe various DC extracts was also determined. In general, apoptosisbegins with a strong milochondrial activation followed by a cellularnuclear degradation. FACS analysis of the above cell cultures enabledalso to calculate the percent apoptosis in each cell culture.

(ii) Results

(a) Effect of DC on the cell cycle of keratinocytes:

As seen in FIGS. 7A and B, and in FIGS. 7E and F, both Narcissus derivedDC and Artemia derived DC had an effect on the DNA content of thekeratinocytes which showed a decrease in percent of cells being in theG1 phase and an increase in cells being in the S and G₂+M phases (theeffect being evident already on day 2 of the incubation and moreapparent on day 5 of the incubation). Against this, as seen in FIGS. 7Cand D, the effect of the Narcussis derived AE (IBR-3) was much lessapparent being slightly evident only 5 days after incubation where adecrease in the percent of cells in the G1 phase was seen with anincrease in the percent of cells being in the S phase and a nonsignificant increase in the percent of cells being in the G₂+M phase.

In addition, the keratinocytes incubated with the Narcissus and Artemiaderived DCs IBR-1 and IBR-4, showed an increase in the percent ofapoptosis while no such increase was seen in keratinocytes incubatedwith Narcissus derived AE IBR-3.

(b) Effect of DC on the cell cycle of fibroblasts

As seen in FIGS. 8A and B and FIGS. 8E and F, Narcissus derived DC(IBR-1) and Artemia derived DC (IBR-4) increased the percent of cellsbeing in the S and G₂+M phases (seen mainly five days after beginning ofincubation) as compared to the same cell cultures incubated with water.Against this, as seen in FIGS. 8C and D, incubation of the cells withNarcussis derived AE (IBR-3) had no effect. None of the tested extractsincreased the percent of apoptosis in the fibroblast cell cultures.

The effect of the various DCs on the keratinocyte and fibroblastcultures was time and dose dependent.

Example XIII Effect of DC Preparations Obtained from Bulbs of VariousPlants on Growth of Germinated Cucumber Seeds

(a) Extracts were prepared from bulbs of various plants as described inExample 1 above. The extracted bulbs were in their dormant stage inwhich no growth tip could be visualized. The extract concentration isdefined as original weight of bulb (gr) per final extract volume (ml).

(b) The effect of extracts on growth of cucumber germinated seeds:

The experimental assay was carried out as explained in Example 1 above.The effect of the tested bulb extracts on the growth of cucumber seedswas tested 24 hours and 48 hours after beginning of the incubation ofthe extracts with the seeds.

The inhibitive effect of the tested extracts was calculated as describedin the Examples above.

Results

As seen in Table 9 below, most of the extracts showed good inhibitoryeffect on the growth of the germinated cucumber seeds (up to about 60%inhibition in average). Several of the plants showed very goodinhibition activity of about 90% inhibition (e.g. Pancratium maritumum).Several of the extracts showed a low inhibitory effect which may, insome cases, be due to the fact that the bulbs were not in fill dormancy.

The effect of the extracts from Pancratium maritumum and HyancinthCarnegie were tested further for their effect on the cucumber seedgrowth by adding various concentrations of the extracts to the seeds.The results (not shown) showed correlation between the concentration ofthe added extract and the inhibition effect of the extract on the growthgerminated cucumber seeds.

TABLE 9 Inhibition (%) of seed growth by extracts: 24 hours 48 hours (%inhibition) (% inhibition) Extract Source Root Hypocotyl Root HypocotylSparaxis 0.52 gr./ml 38 −33 49 39 Hyacinth carnegie 0.40 gr./ml  9 91 9491 Freesia 0.42 gr./ml 62 18 77 7 Crocus 0.41 gr./ml 48 9 30 37Ornithogalum 0.82 gr./ml 52 −18 54 −20 arabicum Montbartia 0.64/gr./ml49 28 63 66 Scilla hyacinthus 1.25 gr./ml 61 0 68 6 Pancratium 0.71gr./ml 90 89 93 96 maritumum (−) indicates growth stimulation

Example XIV Effect of Extracts from Narcissus Bulbs on Growth ofCucumber Plants

(a) Extracts from dormant Narcissus bulbs were prepared as explained inExample 1 above.

(b) Cucumber seeds were germinated and let grow for three days untilthey had roots and the hypocotyl of about 4 cm. The plants were thenplanted in soil and let grow at 23° C. in tap water. The plants weredivided into the following three groups, each comprising 18 plants:

1. Plants that were irrigated with tap water;

2. Plants that were irrigated with tap water and treated with theNarcissus bulb extract by spraying the extract (5 mg/ml) on the leavesand the growth meristem; and

3. Plants which were irrigated directly the Narcissus bulb extract (0.2gr./ml).

The plants were irrigated every day and following one week of treatment,the plants were taken out of the soil and the effect of each treatmentwas tested by measuring the length of the roots and stems of each plant.

Results

As seen in Table 10 below, application of the dormant Narcissus bulbextract on to cucumber plants both by spraying the extract on the leavesand growth meristem (Group 2) as well as by irrigating the plants withthe extract (Group 3) resulted in inhibition of the cucumber plants'growth as compared to their growth with tap water.

TABLE 10 Effect of dormant Narcissus bulb extract on growth of cucumberplants % inhibition Irrigation with: Root Stem (1) Tap water 0 0 (2)Narcissus bulb extract applied on leaves and growth 21% 25% center (3)Narcissus bulb extract applied with irrigation 30% 21%

Example XV Effect of Narcissus Bulb Extract on the Growth of DifferentTypes of Seeds

(a) The dormant Narcissus bulb extract was prepared as explained above.

(b) Several kinds of seeds (tomato, cabbage, melon, water melon, wheat,grass, cucumber, bean, barley, corn and pea) were washed overnight withwater and then let germinate for 24 hrs at 30° C. in the dark on watersoaked filter paper. After 24 hrs, the germinated seeds (20 in eachexperiment set) were applied to a Petri dish with Watman filter papersoaked with the dormant extract.

Each group of seeds was divided into the following groups:

1. Seeds grown in water (control); and

2. Seeds grown with the dormant Narcissus bulb extract.

Various types of seeds were grown with the water or extract (at severalconcentrations) as explained in Example 1 above and followingincubation, the length of the roots and hypocotyls of each seed wasmeasured every 24 hours, depending on the rate of germination andgrowth. The inhibition effect of the extract tested and calculated asexplained above.

As seen in Table 11 below, the dormant Narcissus bulb extracteffectively inhibited the growth of the above seeds. The inhibition wasto different extents.

TABLE 11 Effect of dormant Narcissus on growth of different types ofseeds % Inhibition Seed DC Concentration Roots Hypocotyl Grass 0.02 10072 Water melon 0.02 42 74 Cabbage 0.02 63 82 Cucumber 0.2 93 76 Cucumber0.05 70 72 Cucumber 0.02 52 79 Barley 0.2 84 21 Barley 0.05 81 0 Corn0.2 70 1 Peas 0.2 61 69 Peas 0.05 38 28 Beans 0.2 79 55 Beans 0.05 55 13Beans 0.02 42 5

Example XVI Isolation and Identification of an Active Ingredient in theNarcissus Bulb Extract

Several grams of powder prepared from active and dormant Narcissus bulbswere extracted with acetone methanol (90:10). The extracts separatedusing Thin Layer Chromatography (TLC) techniques. The separation wasconducted on TLC plates (Silica gel 60 F254 from Merck). Runningconditions were water: n-butanol:acetic acid (5:4:1). The detectionmethod was UV light at 254 nm and 365 nm. The bands resulting from theseparation were scraped with the silica from the plate, washed withmethanol and dried at 60° C. The bands appearing in the extracts fromthe active Narcissus bulbs were compared to those in the extracts of thedormant Narcissus bulbs.

Results

Comparison of the bands appearing in the above extract showed that therewas a difference in the expression of two bands (termed “band 4 ” and“band 6”) which appeared at a higher concentration in extracts of thedormant bulbs.

The UV absorption spectrum of the two bands and their inhibitiveactivity on germinated cucumber seeds was tested as explained above.

As seen in FIG. 14, the UV absorption peak of band 4 was at 288 nm andthat of band 6 was at 252 nm. As seen in Table 12 below, bands 4 and 6significantly inhibited the growth of germinated cucumber seeds.

TABLE 12 Band separated by TLC 4 6 UV spectra (nm) in Methanol 288 252Inhibition (%) (2 mg. dry purified compound/ml) 24 hrs Hypocotyl 58 58Root 87 89 48 hours Hypocotyl 60 77 Root 90 94

Example XVII Testing for Toxicity of DC Obtained from Dormant NarcissusBulbs

The toxicity of DC of the invention was tested using the followingmethods::

1. Acute Oral Toxicity (Fixed Dose) Test in Rats The acute oraltoxicological test was based on the protocol, code P/ACU/005, issued atJanuary 1996 by Inveresk Research International (IRI), Tranent, EH332NE, Scotland.

2. Ames Test Salmonella typhimurium mammalian microsome plateincorporation assay was based on Ames B. N., McCann, J., and YamasakiE., Mutation Research, 31:347-364 (1975).

3. Cytoxicity Cytoxicity of the extract (5%) of 0.2 gr/ml extract incosmetic cream, determined by agarose diffusion method was tested byEVIC-CEBA, Bordeaux, France.

4. Irritation potential Irritation potential of the product in cream((5%) of 0.2 gr/ml extract) was determined by means of the HET-CAM test(Chorioallantoic membrane of hen's egg) by EVIC-CEBA, Bordeaux, France.

5. Cutaneous Tolerance Cutaneous tolerance of the extract ((5%) of 0.2gr/ml extract) in cosmetic cream, after repeated application to the skinwas assessed by EVIC-CEBA, Bordeaux, France.

Results

The results of the toxicity testings using the above methods were asfollows:

1. The Acute Oral Toxicity (fixed dose) test in rats oral LD 50>2000mg/kg body weight, has no acute harmful effects on both young female andmale rats.

2. Ames test up to the top-limit dose of 5000 μg/plate, exhibiting noprecipitate and no toxicity, did not induce any mutagenic effects on theAmes test.

3. Irritation potential in cream ((5%) of 0.2 gr/ml extract). Theproduct was found as a normally irritant for this kind of product.

4. Cytotoxicity determination by agarose diffusion method of the productin cream ((5%) of 0.2 gr/ml extract) seems to be low and considered tobe low and considered normal for this kind of product.

5. Cutaneous tolerance—The clinical assessment of cutaneous tolerance ofthe extract in cosmetic cream was tested. The product was found verywell tolerated by the skin.

Example XVIII The effect of DC on Elongation of a Tan (a) The assay

A cream containing 5% dihydroxyacetone (DHA) was administered on to theforearms of an individual. DHA is a compound capable of coloring theupper layers of the skin which is used in self tanning products. Thecream was administered three times until a tan appeared on the forearms.

The tanned area was then divided into the following three parts, eachbeing treated by administration of a different cream twice a day during17 days:

1. Treatment with a cream comprising 5% of the DC IBR-1;

2. A cream identical to the one used in (1) above but which does not:contain IBR-1;

3. A cream comprising alpha hydroxy acid (AHA) (commercially used forskin treatment); and

The amount of color on each skin area was measured using aspectro-colorimeter.

The % effect on elongation of the tan was calculated as:

color of tan of treatment a color of tan after treatment b

color of tan with no treatment color of tan with no treatment×100

Results

The results of the above experiment can be seen in FIG. 10, which showsthe relation between the effect on the duration of the tan of the creamused in 1 above containing the IBR-1 DC as compared to the effect of thecream which contained no IBR-1 (FIGS 10A and B) and the relative effectof the cream used in (3) above (comprising AHA) as compared to no creamat all AHA. (FIGS. 10C and D). As can be seen in FIG. 10A, 5 days afteradministration of the creams, the effect of the cream containing IBR-1on elongation of the tan duration was significantly higher than theeffect of the cream which did not contain IBR-1. The effect was evenmore significant 17 days after administration of the cream as can beseen in FIG. 10B.

FIGS. 10C and D clearly show that the cream used commercially which doesnot contain IBR-1 had a negative effect on the duration of the tan, i.e.their administration resulted in shorter duration of the tan as comparedto no treatment at all. As can also be seen, the cream comprising theAHA shortened the duration of the tan (compared to the duration of thetan with no treatment) 5 days after its administration (FIG. 10C) and 17days after its administration (FIG. 10D).

The above results show that a cream comprising DC of the invention, mostprobably due to its inhibition of proliferation of the skin cells,elongates the duration of a tan. Against this, a cream comprising AHAwhich is commonly used for skin treatment most probably due to itsstimulation of cell division, shortens the duration of the tan.

What is claimed is:
 1. A process for producing a purified dorman extractwhich can inhibit proliferation of target cells or target tissue,comprising: (a) providing producer cells or producer tissue derived froma Narcissus bulb which is xenogeneic to said target cells or targettissue and which is capable of entering into a dormant state; (b)inducing said producer cells or produce tissue to enter into a dormantstate by exposure to high temperature for a sufficient period of time;and (c) conducting a water extraction of the dormant producer cells ordormant producer tissue to obtain a water-soluble dorman extract,whereby the extract is fractionated so as to obtain a purified dormanextract which is capable of passing through a 0.45 uM filter from saiddormant producer cells or dormant producer tissue or from a medium inwhich said dormant cells or tissue was incubated, the dorman extractdisplaying a reversible cell anti-proliferative activity withoutsubstantial cytotoxicity , wherein the activity is substantially higherin said fraction than the same fraction obtained from said producercells or tissue when in non-dormant state.
 2. A process according toclaim 1, wherein said purified dorman extract inhibits the growth ofseedlings.
 3. A process according to claim 2 wherein said purifieddorman extract inhibits growth of cucumber seedlings.
 4. Apharmaceutical or cosmetic composition having an anti-proliferativeeffect on epithelial target cells or tissue of an individual, whichcomprises: the purified dorman extract produced according to the processof claim 1, having an anti-proliferative effect on the epithelial targetcells or tissue of an individual, in combination with a pharmaceuticallyacceptable carrier or cosmetic composition.
 5. A method for inhibitingthe proliferation of target epithelial cells or target epithelial tissuein an individual, comprising administering to the individual aneffective amount of the purified dorman extract prepared according tothe process of claim 1 in a pharmaceutically acceptable composition toinhibit the proliferation of the target epithelial cells or targetepithelial tissue in said individual.
 6. The process of claim 1, wherebythe step of inducing said producer cells or producer tissue to enterinto a dormant state by exposure to high temperature comprises aninitial exposure at a high temperature for a sufficient period of time,followed by a secondary exposure to low humidity in a dry room for amaximal period of 8 months.
 7. A pharmaceutical or cosmetic compositionhaving an anti-proliferative effect on epithelial target cells or tissueof an individual, which comprises: the purified dorman extract producedaccording to the process of claim 6, having an anti-proliferative effecton the epithelial target cells acceptable carrier or cosmeticcomposition.
 8. A method for inhibiting the proliferation of targetepithelial cells or target epithelial tissue in an individual,comprising administering to the individual an effective amount of thepurified dorman extract prepared according to the process of claim 6 ina pharmaceutically acceptable composition to inhibit the proliferationof the target epithelial cells or target epithelial tissue in saidindividual.
 9. The process of claim 1, whereby the step of inducing saidproducer cells or producer tissue to enter into a dormant state, furthercomprises exposure to another factor in addition to high temperature,the factor selected from the group consisting of low humidity, light,growth factors, sugars, and salts.
 10. A pharmaceutical or cosmeticcomposition having an anti-proliferative effect on epithelial targetcells or tissue of an individual, which comprises: the purified dormanextract produced according to the process of claim 9, having ananti-proliferative effect on the epithelial target cells or tissue ofsaid individual, in combination with a pharmaceutically acceptablecarrier or cosmetic composition.
 11. A method for inhibiting theproliferation of target epithelial cells or target epithelial tissue inan individual, comprising administering to the individual an effectiveamount of the purified dorman extract prepared according to the processof claim 9 in a pharmaceutically acceptable composition to inhibit theproliferation of the target epithelial cells or target epithelial tissuein said individual.
 12. The process of claim 1, whereby the step ofinducing said producer cells or producer tissue to enter into a dormantstate by exposure to high temperature comprises exposure to hot water.13. A pharmaceutical or cosmetic composition having ananti-proliferative effect on epithelial target cells or tissue of anindividual, which comprises: the purified dorman extract producedaccording to the process of claim 12, having an anti-proliferativeeffect on the epithelial target cells or tissue of said individual, incombination with a pharmaceutically acceptable carrier or cosmeticcomposition.
 14. A method for inhibiting the proliferation of targetepithelial cells or target epithelial tissue in an individual,comprising administering to the individual an effective amount of thepurified dorman extract prepared according to the process of claim 12 ina pharmaceutically acceptable composition to inhibit the proliferationof the target epithelial cells or target epithelial tissue in saidindividual.
 15. The process of claim 1, wherein the temperature is 45degrees Celsius.
 16. A pharmaceutical or cosmetic composition having ananti-proliferative effect on epithelial target cells or tissue of anindividual, which comprises: the purified dorman extract producedaccording to the process of claim 15, having an anti-proliferativeeffect on the epithelial target cells or tissue of said individual, incombination with a pharmaceutically acceptable carrier or cosmeticcomposition.
 17. A method for inhibiting the proliferation of targetepithelial cells or target epithelial tissue in an individual,comprising administering to the individual an effective amount of thepurified dorman extract prepared according to the process of claim 15 ina pharmaceutically acceptable composition to inhibit the proliferationof the target epithelial cells or target epithelial tissue in saidindividual.
 18. The process of claim 1, whereby the step of inducingsaid producer cells or producer tissue to enter into a dormant state byexposure to high temperature is for a duration of 2 to 4 hours.
 19. Apharmaceutical or cosmetic composition having an anti-proliferativeeffect on epithelial target cells or tissue of an individual, whichcomprises: the purified dorman extract produced according to the processof claim 18, having an anti-proliferative effect on the epithelialtarget cells or tissue of said individual, in combination with apharmaceutically acceptable carrier or cosmetic composition.
 20. Amethod for inhibiting the proliferation of target epithelial cells ortarget epithelial tissue in an individual, comprising administering tothe individual an effective amount of the purified dorman extractprepared according to the process of claim 18 in a pharmaceuticallyacceptable composition to inhibit the proliferation of the targetepithelial cells or target epithelial tissue in said individual.
 21. Theprocess of claim 1, whereby the step of inducing said producer cells orproducer tissue to enter into a dormant state by exposure to hightemperature comprises an initial exposure at a temperature of 45 degreesCelsius for 2 to 4 hours followed by a secondary exposure to lowhumidity in a dry room at 30 degrees Celsius for a maximal period of 8months.
 22. A pharmaceutical or cosmetic composition having ananti-proliferative effect on epithelial target cells or tissue of anindividual, which comprises: the purified dorman extract producedaccording to the process of claim 21, having an anti-proliferativeeffect on the epithelial target cells or tissue of said individual, incombination with a pharmaceutically acceptable carrier or cosmeticcomposition.
 23. A method for inhibiting the proliferation of targetepithelial cells or target epithelial tissue in an individual,comprising administering to the individual an effective amount of thepurified dorman extract prepared according to the process of claim 21 ina pharmaceutically acceptable composition to inhibit the proliferationof the target epithelial cells or target epithelial tissue in saidindividual.